Band 52, Heft 5-6

We report on the preparation of 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide. These vitamin B12-analogs were required as reference compounds for comparison with a corrinoid previously isolated in small amounts from Eu­bacterium limosum grown in the presence of 4(5)-aminoimidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was synthesized from N-1-benzyl-4-nitroimidazole which was reduced to N-1-benzyl-4-aminoimidazole and condensed with 1-dimethylamino-2-methylbutan-3-one to yield N-1-benzyl-4-aza-5,6-dimethylbenzimidazole. The benzyl group of this compound was split off by catalytic hydrogenation to form 4(7)-aza-5,6-dimethylbenz-imidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was transformed by a growing culture of Propionibac­terium shermanii into 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenz-imidazolylcobamide. Both vitamin B 12-analogs were almost as active as Vitamin B12 in a growth test with the vitamin B12-dependent Escherichia coli-mutant DSM 4261.

From the farinose frond exudate of the fern Notholaena rigida two new triterpenes have been identified. They were found to be the C-24 epimers of 12 β-acetoxy-24, 25-diO-isopropylidene-dammarane-3 β, 20(S),24,25-tetra-ol. A survey of all triterpenes found in Notholaena is presented.

Four triterpenoids, the scarce nortriterpenoid platanic acid, two flavonoids and 2,4-dihy-droxyphenylacetic acid methyl ester, hitherto unknown as a natural product, were isolated from Madhuca pasquiery. The structures were established by means of mass and NMR spectroscopy.

A series of Asteraceae, belonging to the tribes Astereae, Eupatorieae, Helenieae and Heli­antheae, have been analyzed for the presence of flavonoid aglycones accumulated on leaf and stem surfaces. Methyl ethers of apigenin, luteolin, kaempferol, and quercetin as well as methyl ethers of their relevant 6 -hydroxy derivatives were abundant, whereas 8 -O-substi-tuted derivatives and flavanones were encountered only occasionally; 6 ,8 -di-O-substituted flavonols were found in only one species.

The influence of ageing on the amount and composition of phenolic compounds and their glucosides was studied in one to five-year-old shoots of Picea abies. The total content of phenolics increased remarkably within the first two years of growth. In this period cinnamic acid derivatives were dominating, but beginning with the end of the first vegetation period a dramatic increase of acetophenones was observed. Obviously at the end of the first vegetation period the cinnamic acid derivatives are subjected to oxidation resulting in an increase of acetophenones.

Pulvinatal (1) was isolated from fermentations of Nidularia pulvinata as an inducer of differentiation of HL -60 promyelocytic leukemia cells. Its structure was elucidated by spectroscopic methods. In addition, N. pulvinata was found to produce 2,4,5-trihydroxy-6-methyl-benzenecarbaldehyde (2) and orsellinic acid (3). Pulvinatal exhibits weak antifungal and only marginal cytotoxic activities.

After growing Pseudomonas aeruginosa in the presence of 2 ,4-[4- 15 N]-diaminobutyric acid (Dab) its incorporation into the quinoline chromophore of the pyoverdin produced by this bacterium could be shown by mass and NMR-spectroscopic techniques. In combination with earlier results it can thus be stated that the precursor of the chromophore is a condensation product of ʟ-Dab and ᴅ-Phe or ᴅ-Tyr. A synthesis for [4 - 15 N ]-Dab is described.

Gassing during 14 days of Aleurites plants with 0.3 ppm SO 2 leads to quantitative modifications of the lipid composition of leaves. The ratio of phospholipids to glycolipids is shifted from 1:3 in control plants to 1:1.8 in SO 2 -plants. Glycolipids decrease from 62% of total lipids in control plants to 50% in SO 2 -plants. On the other hand phospholipids increase from 20% in control plants to 28% of total lipids in SO 2 -plants. This implies that not only the thylakoid membrane but also the mitochondrial membranes and cell membranes of tonoplasts and of the plasmalemma suffer modifications under the influence of SO 2 . With respect to the fatty acid composition, despite the drastic change in the lipid composition no alteration in comparison to control plants is seen. Chlorophylls increase in SO 2 -plants by 24% . Also the content of β-carotene, and of the xanthophylls lutein, violaxanthin and neoxanthin is increased. Only zeaxanthin exhibits a decrease. From the changes in the chlorophyll and carotenoid content it can be concluded that in SO 2 -plants the light-harvesting-complex (LHCP) is stronger developed (P. He, A. Radunz, K. P. Bader and G. H. Schmid, Z. f. Naturforsch. 51c, 441- 453 and 833-840, 1996).

Phenylacetic acid (PA), a phytotoxic product of the bacteria accompanying the virulent nematode isolate OKD-3 was detected in the callus of Pinus thunbergii after inoculation with the nematode. The amount of PA detected was large enough to induce the formation and accumulation of benzoic acid (BA) and its conjugates in the callus. These results further support the hypotheses that PA is the pathogenic toxin and that the PA-producing bacterial strains accompanying the pathogenic nematode are the genuine pathogens of the pine wilt disease.

General (polygenic) resistance of plant hosts to an attack by a range of pathogens is an important feature of plant defense responses against the infection. In search of biochemical markers defining this resistance, cell suspensions derived from leaves of potato (Solanum tuberosum L .) cvs. Tarpan and Bzura that are polygenically susceptible and resistant to Phytophthora infestans, respectively, were inoculated with culture filtrate (CF) of the fungus. Cell suspension of Tarpan responded to CF treatment by a higher extracellular alkalinization and more significant reduction in their viability and growth than those of the Bzura cultivar. The stimulation of phenylalanine ammonia-lyase (PAL ) activity but not of β-1,3-glucanase, was significantly higher in CF treated Bzura cells than in Tarpan ones. The obtained results suggest that sensitivity to the fungal toxins and variation of PAL activity may represent useful markers for the evaluation of polygenic resistance in potato.

Flowers of A. odorata yielded six insecticidal rocaglamide derivatives including four compounds which proved to be new natural products. Structure elucidation of the new com pounds, including establishment of their absolute configurations by CD spectroscopy and NMR is described. When incorporated into artificial diet all isolated rocaglamide derivatives exhibited strong insecticidal activity towards neonate larvae of the polyphagous pest insect Spodoptera littoralis with LC 50 s varying from 1.5-53.4 ppm (2.9-97.1 nᴍ) . Two of the isolated compounds which showed LC 50 s of 1.5 and 1.6 ppm (2.9 and 3.2 nᴍ ) respectively are similar with regard to their insecticidal activity to azadirachtin (LC 50 0.9 ppm [1.3 nᴍ] ) which was included into the study as a positive control.

Peroxidizing herbicides, as inhibitors of protoporphyrinogen IX oxidase (protox), cause accumulation of protoporphyrin IX and lead to a rapid phytotoxic degradation of plant cell membranes in the light. Certain 5-arylimino-3,4-tetramethylene-1,3,4-thiadiazolidin-2-ones are peroxidizing compounds. They are isomerized to the corresponding more active triazolidines with glutathione S-transferase (GST) in the presence of reduced glutathione (GSH). The rate of isomerization depends on the arylimino moiety and the presence of carbonyl or thiocarbonyl groups at the thiadiazolidine ring. The following compounds and their isomeric triazolidines were synthesized: 5-(4-Bromophenyl)-3,4-dialkyl-1,3,4-thiadiazolidin-2-ones (I), 5-(4-brom ophenyl)-3,4-dialkyl-1,3,4-thiadiazolidine-2-thiones (II), 5-(4-brom ophenyl)-3,4-alkylene-1,3,4-thiadiazolidin-2-ones (III), 5-(4-brom ophenyl)-3,4-alkylene-1,3,4-thiadiazolidine-2 -thiones (IV). The phytotoxic activities of these compounds were investigated using Scenedesmus acutus and protoporphyrinogen IX oxidase isolated from corn (Zea mays) etio­plasts. These compounds are peroxidizing compounds, and the triazolidines showed higher activity than the thiadiazolidines. Compounds I and III were isomerized to the corresponding triazolidines, while compounds II and IV were not or scarcely converted into their isomers with GST in the presence of GSH . Seemingly, the bulkiness of alkylene or dialkyl groups at the heterocycle influences the isomerization of carbonyl-substituted thiadiazolidines.

The results presented in this paper indicate that procyclic forms of Trypanosoma brucei possess a phosphatase activity detected in the external cell surface able to hydrolyze about 0.7 nmol ∙ mg −1. min −1 p-nitrophenylphosphate. A faster rate of hydrolysis was observed when membrane-enriched fractions were used. This activity is weakly sensitive to 1 mᴍ NaF, 10 mᴍ tartrate and 10 mᴍ levamizole but strongly inhibited by 0.1 mᴍ vanadate. Inhibition by both NaF and vanadate have a competitive character. This phosphatase activity decreases by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained during at least 1 hour. In the membrane-enriched fractions this phosphatase activity showed to be an acid phosphatase. In addition, intact cells could catalyze the dephosphorylation of [ 32 P]phosphocasein phosphorylated at serine and threonine residues.

The effects of yeast extract (0-10g/l), methanol (0-10%v/v), acetic acid (0-1.0g/l), furfural (0-0.5g/l), glucose (0-30g/l), inoculum age (15-70h) and product concentration (18-230g/l) on the xylose-xylitol conversion by Candida guilliermondii FTI 20037 were studied. The xylitol specific productivity increased about 35% at a yeast extract concentration of 1.0g/l, whereas glucose showed a strong inhibitory effect on the xylitol production and a stimulating effect on the growth of C. guilliermondii. Methanol, acetic acid and furfural under the employed concentrations did not show any positive effect neither on the growth or on the xylose-xylitol conversion by the yeast. The inoculum age showed a strong influence on xylitol formation and the best fermentative parameters were attained using a 40-h inoculum age. A xylitol concentration in the fermentation medium higher than 80g/l inhibited mark­edly the xylitol productivity by the yeast C. guilliermondii.

The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo. For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNA-layer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics. Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 ± 0 .9)× 10 −11 breaks/Gy × dalton. The ratio of ssb/dsb was 0.5 ± 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

The detection of cGMP in yeast (Eckstein 1988), but lacking hints at guanylate cyclase from sequencing of the yeast genome, raised questions about existence, isoform, and regula­tion of guanylate cyclase from this organism. We found a particulate guanylate cyclase activity in yeast extracts, exhibiting properties of an integral membrane protein. Characteristics are: pH-optimum at pH 6.8, temperature-optimum around 60 °C, only slight stimulation by Mn 2+ . Sigmoidal enzyme kinetics indicate allosteric regulation, ATP and Ca 2+ act as negative allosteric effectors. The enzyme activity is increased by yeast alpha-1 mating factor, and by sodium nitrite, thus showing properties of particulate as well as of soluble isoforms from other eukaryotes. The activation by alpha-1 mating factor suggests receptor functions, and a role in ascospore conjugation.

Comparative kinetic studies on the oxidation of catecholamines (CA) (dopamine (DA), epinephrine (EP). norepinephrine (NEP)) serving as a neuromediator in the sympathetic nervous system, 3,4-dihydroxyphenylalanine (DOPA) and 6-hydroxydopamine (6-OHDA), a wellknown neurotoxic agent, were performed in the presence of ascorbate (AscH - ) in 50 mᴍ phosphate buffer, pH 7.40, at 37 °C by using a Clark electrode, EPR and the absorption spectroscopy. The oxidation of CA and DOPA alone was found to be a self-accelerating process, with quinone products (Q) acting as autocatalysts. The rate of oxygen consumption (R ox ) increased with time and reached a steady-state level. A starting value of R ox increased in the order: EP < DOPA ≈ NEP ≪ DA ≪ 6-OHDA, whereas a steady-state value of R ox changed in the order: DOPA < DA < NEP ≪ EP ≪ 6-OHDA. The changes in R ox with time were found to correlate with the resistance of primary Q to the intramolecular cyclization. The effect of AscH - on CA oxidation depended dramatically on whether AscH - was added to non-oxidized or preoxidized CA. Added to non-oxidized CA and DOPA, AscH - inhibited their oxidation (but not that of 6-OHDA). For the case of DA, a pronounced lag period was observed by both a Clark electrode and spectrophotometrically. The addition of AscH - to preoxidized CA, DOPA and 6-OHDA induced an increase in R ox a steadystate concentration of the ascorbyl radical. The kinetic behaviour of the systems was determined by two major factors: 1) AscH - suppressed the formation of Q, a catalyst for CA oxidation, most likely due to the reaction of AscH - with the semiquinone formed from CA; 2) Q derived both from CA and 6-OHDA catalyzed AscH - oxidation. The elevated cytotoxicity of 6-OHDA was found to be in part caused by the condition that 6-OHDA oxidation was not inhibited by AscH - and by the high efficiency of 6-OHDA as a redox cycling agent in combination with A scH − . These observations explain the very pronounced and prolonged cytotoxicity of 6-OHDA even at low concentrations that increases at elevated concentrations of AscH - .

Three Hungarian (No.2, 4 and 9), and a Moldavian (K) plum pox virus isolates were compared with a characterized Spanish isolate (5.15) by RT-PCR, ELISA, dot-blot and West­ern blot analysis. Monoclonal antibodies prepared against the external, intermediate and internal sequences of the coat protein of the Spanish isolate were able to differentiate the four isolates. Hungarian isolate No. 2 proved to be serologically identical to the Spanish isolate, while No. 4 showed appreciable differences and No. 9 could be recognized only by the monoclonal antibodies representing the intermedial and internal parts of the coat protein. K isolate showed a more distant relationship to other isolates. Our experiment provided the first demonstration of the presence of D type isolates in Hungary.

To estimate the necessity of air ions (AIs) as a natural source of atmospheric gaseous superoxide for mammalia the effect of air ion deprivation on mice and rats was investigated. Ambient air deionization inside an experimental acrylic glass cage (AGC) was performed by electrostatic field, built up by acrylic glass surfaces. Under these conditions, four hours after the animals were placed into the AGC, the concentration of negative AIs was not detectable, the concentration of positive ions was (mean ± SD) 77 ± 18 ionsxcm -3 . The negative and positive AI concentrations in identical silicate glass cages (control) were 482 ± 128 ionsxcm -3 and 660 ± 148 ionsxcm -3 respectively. It was found that the prolonged deficiency of AIs in ambient air leads to accelerated death of the animals. The life span of the deprived mice and rats was 16.2 ± 0.9 and 23.0 ± 1.1 days respectively. The pathological symptoms and ultrastructural changes in the adeno-and neurohypophysis in deprived animals observed strongly suggest that animal death is related to disturbances in neurohormonal regulation and pituitary insufficiency. The possible physiological need of AIs and atmospheric superox­ide, and its role in the development of environmental stress in human beings and in particular in premature infants is discussed.

The effect of the anticancer drug cisplatin (alone and in combination with γ-radiation) on the initiation of DNA synthesis in Friend leukemia cells was studied. A method for isolation of DNA fractions containing the origins of replication was used. It was found that cisplatin decreased the rate of the initiation of DNA synthesis. The mild γ-radiation has previously been observed to inhibit the initiation of DNA synthesis. In the present investigation the combination of cisplatin and γ-radiation showed additive effects without synergism on the initiation of DNA biosynthesis.

Treatment of chicken blood smears and semithin sec­tions from Epon-embedded mouse tissues with aqueous solutions of the 3-aminonaphthalimides FA-142, FA-2043, and FA-2143 induced a strong green-yellow fluorescence of chromatin under violet or violet-blue excitation. Chromatin emission was abolished by previous DNase or hot TCA treatment. The use of 3-methoxy (FA-655) and 3-nitro derivatives (M-4212 and M-12210) resulted in very weak fluorescence of chromatin. Ab­sorption maxima at 346 and 408 nm and an emission peak at 570 nm were observed for the free compound FA-142. Fluorescence properties open new and interesting applications for some of these antitumoral and DNA-intercalating naphthalimides.